EXCEED THE SPACE PROVIDED. The long term goal of this research is to determine the role of autoinduction (quorum sensing (QS)) in the pathogenesis of Pseudomonas aeruginosa. In P. aeruginosa, two QS systems, las and rhl, plus a third signal (Pseudomonas quinolone signal) (PQS) control the expression of many virulence factors and group behavior such as biofilm development, swarming and twitching. Recent studies, including transcriptome analysis, indicates that the P. aeruginosa QS regulon is much more complex than previously thought. Our hypothesis is that the QS regulon is composed of a complex regulatory cascade with the principal regulator LasR-3O- C12 HSL regulating expression of the genes qscR, vqsR and rhlR. These later genes are also transcriptional regulators (TRs) and we refer to them and lasR as the master regulators. As a group these four master TRs control expression of 33 known or putative TRs and over 550 other genes. The specific aims of the current R37 extension propos'al are: 1. Determine which genes are regulated by RhlR-C4 HSL and VqsR. (Others are working on qscR). 2. Determine which of the TRs are involved in P. aeruginosa virulence and biofilm development. 3. Further characterize TR genes identified in aim 2. Use unmarked, null mutants in traditional biofilm models as well as a novel biofilm model employing airway surface liquid (ASL) and in two models of P. aeruginosa infection (nematodes and leaf lettuce). Do transcriptome analysis on selected TR null mutants and their complemented versions. 4. Initiate studies on the role of QS in mixed cultures of P. aeruginosa and Staphylococcus aureus or Burkholderia cenocepacia. When wild type strains of these organisms are mixed together in planktonic cultures, the P aeruginosa takes over. We will determine if that requires QS regulated genes and if this dominance is also seen in biofilms. The results of these studies will further our understanding of regulation of virulence in P. aeruginosa. They will provide new insights into quorum sensing systems in general and determine the feasibility of a new approach to therapy for gram negative infections.